A variety of methods are currently employed in molecular biology and clinical diagnostic to identify polymorphisms (e.g., single nucleotide polymorphisms (SNPs) and mutations) in DNA or RNA. Such methods include kinetic PCR, microarrays, RNA interference, antisense inhibition or nanosensors. Discrimination between polymorphic variations typically relies upon detection systems based on melting temperature differences between perfectly matched and mismatched duplexes relative to a hybridizing probe. The utility of such hybridization-based methods, however, is limited because the differences in melting temperature are typically too small to discriminate single mismatches within probes. Although shorter probes can be used to improve detection of a mismatch, they sacrifice specificity for discrimination when used in complex nucleic acid samples.
Recently, chimeric probes having select positions modified with locked nucleic acids (LNAs) have been reported to enhance both duplex stability and mismatch discrimination. LNA monomers contain a modified ribose moiety and are somewhat similar to 2′-O-methyl RNA, except that the O-methyl group bridges and constrains the 2′ and 4′ carbons of the ribose ring. This covalent bridge effectively ‘locks’ the ribose in the N-type (3′-endo) conformation that is dominant in A-form DNA and RNA. This locked conformation enhances base stacking and phosphate backbone pre-organization and significantly increases the thermal stability of the oligonucleotide, resulting in improved affinity for complementary DNA or RNA sequences (higher Tm).
The enhanced affinity of LNA nucleotides increases the sensitivity and specificity of expression analysis in cDNA microarrays, FISH probes, real-time PCR probes and other molecular biology techniques based on oligonucleotide hybridization. For example, chimeric oligonucleotides incorporating LNA units have been shown to be useful for allele-specific PCR detection of polymorphisms. Johnson et al. (Nucleic Acids Research, 2004, Vol. 32, No. 6 e55) disclose LNA oligonucleotides for allele-specific PCR (AS-PCR), with LNA added throughout at several positions in the primer. Chou et al. (BioTechniques 39:644-650 (November 2005)) disclose LNA oligos with the LNA at the −5 position. Latorra et al., Human Mutation 22:79-85 (2003) and Latorra et al., BioTechniques 34:1150-1158 (June 2003), also disclose LNA oligos with the LNA at the 3′ end of the primer. You et al., Nucleic Acids Research, 2006, Vol. 34, No. 8 e60, disclose LNA oligonucleotides for detecting SNPs based on melting curve analysis. Incorporating deliberate mismatches into primers for AS-PCR is another technique which has been used to improve discrimination. For example, Song et al., AAPS PharmSci 2002; 4 (4) article 29, designed primers that incorporate a deliberate mismatch at the −3 position.
The present invention is directed to oligonucleotides that demonstrate improved ability to discriminate between alleles on the basis of single nucleotide polymorphisms.